The most important medical challenges include cardiovascular diseases, stroke, degenerative neurological diseases, diabetes, arthritis, osteoporosis, kidney and liver failure, spinal cord injury, burns, battlefield trauma, and other devastating conditions. Organ transplantation addresses some of these needs, but the scarcity of donors and the risk of immune suppression pose major limitations on transplantation. Regenerative medicine seeks to devise new ways to repair or replace damaged tissues and organs for millions of patients who cannot receive transplants. A core technology is the bioengineering of a functional tissue or organ by seeding living cells onto a biodegradable scaffold and then surgically implanting the construct into a patient. Tissue engineering involves extensive remodeling of cells and scaffolds. A major barrier to progress has been the inability to monitor this dynamic complex biological process in real-time, which makes control and optimization extremely difficult. On the other hand, as defined in the NIH roadmap molecular imaging plays an increasingly important role in the advancement of medicine. The optical molecular imaging tools has now allowed much better understanding of biological interactions at molecular and cellular levels in mouse models of almost all human diseases, and found several major clinical applications. Therefore, we are motivated to integrate these two forefront technologies in biomedical research - tissue engineering and optical molecular imaging - in a single unified framework, and drive a paradigm shift from static assays of cellular function in biopsied tissue or 2D culture models towards systematic analysis of 3D systems. The overall goal of this project is to develop a first-of-its-kind multi-probe multi-modal optical molecular tomography system for regenerative medicine and to demonstrate its utility in assessing the bioengineered blood vessels at the pre- and post-implantation stages. Fluorescent probes will be used to label the tubular scaffold and the two main cell types of blood vessels (endothelial cells lining the lumen, and smooth muscle cells in the wall). Optical fibers embedded within the scaffold will deliver laser light for optical coherence tomography and to excite the fluorescent probes. Innovative algorithms will be developed to reconstruct 3D distributions of multiple fluorescent probes. The proposed imaging system will first be used to track the development of bioengineered vessels in 100m resolution in a bioreactor mimicking blood flow conditions. Additional fluorescent probes will be used to monitor cell-specific gene expression and verify physiological responses of cells within the engineered vessel. The vessels will then be implanted as interposition grafts in the carotid arteries of living sheep, and will be imaged in 500m resolution to follow the tissue regeneration and function. Successful completion of the project will create new optical molecular imaging tools with a demonstrated application in vessel engineering, and have major and lasting impacts on many other areas in regenerative medicine.